1. Using the concentration of your sample calculate the volume of DNA solution needed to obtain your target mass of DNA (typically between 30-50μg).  The water volume will be determined by how much is needed for the reaction mixture to reach 50 µL.

2. Add reagents: 

2 µL MBG water

42 µL 774 ng/µL CFTR_pGHE DNA (32.51 µg DNA)

5 µL 10X FD buffer (enzyme rack, FreezerA)

1 µL FD Nhe1 restriction enzyme **(incubate overnight for Nhe1, but for FD Nhe1 (FastDigest), do 2 hours)

3. Obtain a 1.5 mL tube and label with the name of the DNA plus LIN. Add the reagents following the order in the table above. Incubate for 2 hours at 37°C to ensure complete digestion. Move to temporary rack in Freezer A.

08/02/2022

1. Using the concentration of your sample calculate the volume of DNA solution needed to obtain your target mass of DNA (typically between 30-50μg).  The water volume will be determined by how much is needed for the reaction mixture to reach 50 µL.

2. Add reagents: 

42.7 µL MBG water

1.3 µL 774 ng/µL CFTR_pGHE DNA (1 µg DNA)

5 µL 10X FD buffer (enzyme rack, FreezerA)

1 µL FD Nhe1 restriction enzyme **(incubate overnight for Nhe1, but for FD Nhe1 (FastDigest), do 2 hours)

3. Obtain a 1.5 mL tube and label with the name of the DNA plus LIN. Add the reagents following the order in the table above. Incubate for 2 hours at 37°C to ensure complete digestion. Move to temporary rack in Freezer A.

**DO NOT USE THIS UNLESS SPECIFICALLY ASKED TO DO SO

HighYield T7 ARCA mRNA Synthesis Kit

NOTE: Take extra care in preparing RNA. Wear gloves and put on Eliminase as samples are easily contaminated. 

1. Obtain a 1.5 mL tube and label with the name of the DNA plus RNA. Determine amount of template DNA necessary

2. Place HighYield T7 RNA Polymerase Mix on ice. 

3. Thaw all other reagents to RT, then vortex and spin down. 

4. Add the following reagents to your labeled 1.5 mL tube in order: 

4.92 µL PCR-grade water (based on amount of Template DNA required to get to 20 µL; determine the amount of template DNA required before beginning RNA synthesis) 

2 µL HighYield T7 Reaction Buffer 10X

2 µL DTT

Vortex and spin down

1.2 µL ARCA

0.3 µL GTP 

1.5 µL N1-Methylpseudo-UTP 

1.5 µL CTP

1.5 µL ATP 

3.08 µL Template DNA (want 1-8 µg DNA) 

Vortex and spin down

2 µL HighYield T7 RNA Polymerase Mix 

Vortex and spin down

TOTAL VOLUME: 20 µL

5. Incubate at 37ºC for two hours. 

PolyA Tailing (different kit in Freezer B) 

1. Add the following reagents into the RNA tube(s) and incubate at 37°C for 1 hour.

**NOTE: Do NOT use regular MBG water from Fridge A! Either use what is in the kit or if there is not any in the kit, use the RNA MBG water in Freezer C Box 13. 

36 µL Nuclease Free Water

20 µL 5X E-PAP Buffer

10 µL 25 mM MnCl2

10 µL ATP Solution

4 µL E-PAP Enzyme

Incubate at 37ºC for 1 hour. 

Stored in -80ºC Freezer in CFTR RNA Box

HighYield T7 ARCA mRNA Synthesis Kit

NOTE: Take extra care in preparing RNA. Wear gloves and put on Eliminase as samples are easily contaminated. 

1. Obtain a 1.5 mL tube and label with the name of the DNA plus RNA. Determine amount of template DNA necessary

2. Place HighYield T7 RNA Polymerase Mix on ice. 

3. Thaw all other reagents to RT, then vortex and spin down. 

4. Add the following reagents to your labeled 1.5 mL tube in order: 

4.92 µL PCR-grade water (based on amount of Template DNA required to get to 20 µL; determine the amount of template DNA required before beginning RNA synthesis) 

2 µL HighYield T7 Reaction Buffer 10X

2 µL DTT

Vortex and spin down

1.2 µL ARCA

0.3 µL GTP 

1.5 µL UTP 

1.5 µL CTP

1.5 µL ATP 

3.08 µL Template DNA (want 1-8 µg DNA) 

Vortex and spin down

2 µL HighYield T7 RNA Polymerase Mix 

Vortex and spin down

TOTAL VOLUME: 20 µL

5. Incubate at 37ºC for two hours. 

PolyA Tailing (different kit in Freezer B) 

1. Add the following reagents into the RNA tube(s) and incubate at 37°C for 1 hour.

**NOTE: Do NOT use regular MBG water from Fridge A! Either use what is in the kit or if there is not any in the kit, use the RNA MBG water in Freezer C Box 13. 

36 µL Nuclease Free Water

20 µL 5X E-PAP Buffer

10 µL 25 mM MnCl2

10 µL ATP Solution

4 µL E-PAP Enzyme

Incubate at 37ºC for 1 hour. 

Stored in -80º Freezer in CFTR RNA Box

1. Gather desired RNA samples and record the sample order and RNA volume added as explained in the DNA sample preparation protocol.

2. For each RNA sample, add: 

1 µL of RNA

2 µL of MBG water (Fridge A)

3 µL of 2X RNA Dye (Box 13, Freezer C)

3. The RNA ladder requires additional preparatory steps before it can be loaded.

a.       Combine 2 μL of ssRNA Ladder (Box 13, Freezer C) with 8 μL of 2X RNA Dye.

b.       Obtain a heating block and thermometer (both in Drawer B). Place the thermometer in the smallest hole and turn the ON/OFF switch to high. 

c.        Turn the high temperature control dial up to increase the temperature. Adjust the temperature according to the following settings: Incubate at 90°C for 2 minutes. 

d.       Immediately place it on ice for 2 minutes and load the entire volume into its corresponding well.

4. When the gel has set, it will have a certain glimmer to it and look solid. Lift the gel tray up and out of the tank. Turn the gel around to where the rubber gaskets are not touching the tank walls.  Fill the electrophoresis tank with 0.5X TBE until the liquid level is the same in both sides of the tank and the gel is covered in TBE.

5. Spin the samples down before loading them. Set your pipette to ~6.5 µL to ensure complete transfer of the samples. Load samples according to the written order in the notebook. For the RNA ladder, load the entire volume into the well. Do not pierce the gel.

6. Slide the tank cover on ensuring that the negative prod is on the same side as the DNA. Turn the electrophoresis machine on by flipping the switch on the front. Run the gel for 90 minutes at 60 V.

RNA Ladder: RiboRuler (SM1823, Thermo Scientific)

If you load 4 µL of RiboRuler you get the following amounts in the bands. You may need to load 8 µL or more to see clear bands.

1. Add 110 µL of acidic PCl (pH 4.5, Fridge B) from the bottom layer. Vortex for 10 seconds and centrifuge at 13,000 rpm for 1 minute.

2. Transfer the top layer to a new, previously labeled 1.5 mL tube. Add 110 µL of chloroform (Fridge A). Vortex for 10 seconds and centrifuge at 13,000 g for 1 minute.

3. Transfer the top layer to a new, previously labeled 1.5 mL tube. Add 110 µL of isopropanol (Fridge A) and place in -20°C freezer (any normal freezer) for 30 minutes.

4. Centrifuge the tubes for 15 minutes at 13,000 rpm and 4°C. Use the centrifuge in the cold room on the 3rd floor.

Did this twice (12 minutes the second time)

5. Carefully decant supernatant down the drain by tilting the tube(s) until it is completely upside down. Add 500 µL of 70% ethanol (Fridge A) and centrifuge at 13,000 rpm for 1 minute.

Two beautiful pellets!

6. Carefully decant supernatant down the drain by tilting the tube(s) until it is completely upside down. Touch the tube to a Kimwipe to wick away any residual ethanol. Set the tube(s) on a rack, open the cap, and set a Kimwipe on top.

7. Transfer phenol and chloroform wastes from all tubes into the appropriate waste bottle. 

8. Re-suspend the dry RNA in 15 µL of MBG water (part of the same RNA kit, Freezer B).

Please put the RNA tube in an appropriate box in the -80 freezer (2nd floor).

CFTR RNA Box